1. Field of the Invention
The present invention relates to methods of producing polypeptides in a mutant of a Bacillus cell, methods of obtaining the mutants of Bacillus cells, and the mutants of Bacillus cells.
2. Description of the Related Art
Surfactin is a cyclic lipopeptide with remarkable surfactant properties which is produced primarily during the stationary phase of growth by several species of Bacillus (Carswell et al., 1994, Applied Microbiology and Biotechnology 41: 281-285; Lin et al., 1994, Applied and Environmental Microbiology 60: 31-38; Morikawa et al., 1992, Journal of Fermentation and Bioenginnering 74: 255-261; Arima et al., 1968, Biochemical Biophysical Research Communications 31: 488-494). The lipopeptide contains seven amino acids, L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu (SEQ ID NO:35) linked to 3-hydroxy-13-methyl-tetradecanoic acid through an amide bond between the carboxy group of the fatty acid and the amino group of glutamic acid and an ester bond between the carboxyl group of the last Leu and the hydroxyl group of the fatty acid. A homologous series with lipidic chainlengths of 13, 14, and 15 carbons (Hosono and Suzuki, 1983, Journal of Antibiotics 36: 667-673; Razafindralambo et al., 1993, Journal of Chromatography 639: 81-85), and isoforms named Val7!-, Ile7!-, and Ala4!-surfactin differing by the seventh or the fourth amino acid (Peypoux et al., 1994, European Journal of Biochemistry 224: 89-96; Baugmart et al., 1991, Biochemical Biophysical Research Communications 177: 998-1005) are known.
A multienzyme complex encoded by the srf operon is reportedly responsible for the nonribosomal biosynthesis of surfactin via the so-called thiotemplate mechanism. The operon contains at least four genes, srfA, srfB, srfC, and srfD. The genes srfA, srfB, srfC, and srfD were previously known as srfAA, srfAB, srfAC, and srfAD, respectively. SrfA, srfB and srfC encode the surfactin synthetase subunits, each containing one or more amino acid-activating domains necessary for the activation of the surfactin substrate amino acids to produce surfactin (van Sinderen et al., 1993, Molecular Microbiology 8: 833-841; Nakano and Zuber, 1989, Journal of Bacteriology 8: 821-831; Cosmina et al., 1993, Molecular Microbiology 8:821-831). The multienzyme complex is organized in seven large domains clustered on three separate proteins (Menkhaus et al., 1993, Journal of Biological Chemistry 268: 7678-7684; Gulli et al., Biochimica et Biophysica Acta 1205: 19-28). The seven domains are responsible for the activation and binding of the seven amino acids of surfactin. According to the thiotemplate mechanism, the adenylation and binding of a specific amino acid take place at the corresponding amino acid-activating domain, a process which requires the cofactor 4-phosphopantetheine. Subsequent trans-thioesterification reactions result in a growing peptide chain, the order of which is determined by the spatial arrangement of the multienzyme subunits. It is currently not known how and when the fatty acid moiety is linked to the peptide and how the ester bond is formed to make the molecule cyclic. Furthermore, the gene sfp is thought to be involved in expression (secretion) of surfactin (Nakano et al., 1992, Molecular General Genetics 232: 313-323).
Bacilli are well established as host cell systems for the production of native and recombinant proteins. However, Bacillus hosts with the desirable traits of increased protein expression and secretion may not necessarily have the most desirable characteristics for successful fermentation. Specifically, the fermentation may not be optimal because of an increase in foaming as biomass increases. Increased foaming limits the productivity of the fermentation.
It is therefore an object of the present invention to provide improved Bacillus hosts which combine the capacity for expression of commercial quantities of protein with satisfactory fermentation characteristics, such as rapid growth and low foaming, thereby enhancing fermentative productivity.